Investigation of Substrate Activation by 4-Chlorobenzoyl-Coenzyme A Dehalogenase

Taylor, Katherine A.; Hong, Xueming; Liu, Rui‐Qin; Yang, Guang; Dunaway‐Mariano, Debra

doi:10.1021/bi962765i

Cited by 33 publications

(56 citation statements)

References 29 publications

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“…The aromatic groups around the benzoyl ring likely assist in the polarization of the -electrons in the benzoyl ring during the reaction (11,12). Mutation studies of this enzyme by Dunaway-Mariano and coworkers (13) have shown that Trp-137, which forms a hydrogen bond with the catalytic Asp-145, has a significant effect on the formation of the Meisenheimer intermediate. Although the overall catalytic constant, k cat , for the conversion of 4-CBA-CoA to 4-HBACoA is reduced only Ϸ60-fold when Trp-137 is mutated into a phenylalanine (13), the rate of forming the Meisenheimer intermediate is reduced 20,000-fold for this mutant (D. Dunaway-Mariano, personal communication).…”

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confidence: 99%

“…Mutation studies of this enzyme by Dunaway-Mariano and coworkers (13) have shown that Trp-137, which forms a hydrogen bond with the catalytic Asp-145, has a significant effect on the formation of the Meisenheimer intermediate. Although the overall catalytic constant, k cat , for the conversion of 4-CBA-CoA to 4-HBACoA is reduced only Ϸ60-fold when Trp-137 is mutated into a phenylalanine (13), the rate of forming the Meisenheimer intermediate is reduced 20,000-fold for this mutant (D. Dunaway-Mariano, personal communication). Comparison of the crystal structures of the wild-type (WT) and W137F mutant enzymes shows that the side chain of Asp-145 has reoriented in the W137F enzyme to be pointing almost 180°from the direction in the WT.…”

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confidence: 99%

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The active site dynamics of 4-chlorobenzoyl-CoA dehalogenase

Lau

Bruice

2001

Proc. Natl. Acad. Sci. U.S.A.

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A molecular dynamics study was performed to compare the differences in the active-site dynamics of the wild-type and W137F mutant enzymes of 4-chlorobenzoyl-CoA dehalogenase. Only in the wild-type simulation are conformations formed between the catalytic Asp-145 and 4-chlorobenzoyl-CoA, which resemble the ab initio calculated gas-phase transition-state geometry. In the W137F simulation, the hydrogen bond formed between His-90 and Asp-145 persisted throughout the simulation, causing the carboxylate of Asp-145 to be distant from the benzoyl ring of 4-chlorobenzoylCoA. In both simulations, water molecules were able to diffuse into the active site of the enzymes. The trajectories provide insight into the routes that water may use to get into position for the hydrolysis portion of the dehalogenation reaction. In both simulations, the water molecule entering the active site forms a hydrogen bond with Asp-145.

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confidence: 99%

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confidence: 99%

The active site dynamics of 4-chlorobenzoyl-CoA dehalogenase

Lau

Bruice

2001

Proc. Natl. Acad. Sci. U.S.A.

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“…The spectroscopic data showed that electron-polarizing forces can be brought to bear in the active site, which, in the case of the bound product, cause a major rearrangement of the electrons in the benzoyl moiety. Interest in these findings has recently been heightened by the publication of the structure of the dehalogenase-4-HBACoA complex derived by X-ray crystallography (7), and the X-ray results have been used to analyze shifts in absorption maxima for ligand-enzyme complexes involving several site-selected mutants (8). Recently, we published an analysis of the polarizing forces in the active site, combining Raman spectroscopic data on the bound product, including results on 13 C and 18 O substituted isotopomers of the product, with structural conclusions culled from the X-ray crystallographic studies (9).…”

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confidence: 99%

Modulating Electron Density in the Bound Product, 4-Hydroxybenzoyl-CoA, by Mutations in 4-Chlorobenzoyl-CoA Dehalogenase Near the 4-Hydroxy Group

et al. 1999

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The enzyme 4-chlorobenzoyl-CoA dehalogenase hydrolyzes 4-chlorobenzoyl-CoA (4-CBACoA) to 4-hydroxybenzoyl-CoA (4-HBA-CoA). Biochemical and crystallographic studies have identified a critical role for the dehalogenase residue Asp 145 in close proximity to the ligand's 4-hydroxy group in the structure of the product-enzyme complex. In the present study the effects of site selective mutations at Asp 145 on the product complex are explored by Raman spectroscopy. The spectral signatures of the WT-product complex, the large red shift in λ max , and the complete reorganization of the benzoyl ring modes in Raman data are absent for the D145E complex. The major spectral perturbations in the WT complex are brought about by strong electron "pull" at the benzoyl carbonyl and electron "push" by the side chain of Asp 145 near the 4-OH group. Acting in concert, these factors polarize the benzoyl's π-electrons. Since the Raman data show that very strong electron pull occurs at the benzoyl's carbonyl in the D145E complex, it is apparent that the needed electron push near the benzoyl's 4-OH group is missing. Thus, very precise positioning of Asp 145's side chain near the benzoyl's 4-position is needed to bring about the dramatic electron reorganization seen in the WT complex, and this criterion cannot be met by the glutamate side chain with its additional CH 2 group. For two other Asp145 mutants D145A and D145S that lack catalytic activity, Raman difference spectroscopic data for product complexes demonstrate the presence of a population of ionized product (i.e., 4-O -) in the active sites. The presence of the ionized phenolate form explains the observation that these complexes have highly red-shifted absorbance maxima with λ maxs near 400 nm. For the WT complex only the 4-OH form is seen, ionization being energetically expensive with the presence of the proximal negative charge on the Asp 145 side chain. Semiquantitative estimates of the pK a for the bound product in D145S and D145A indicate that this ionization lies in the pH 6.5-7.0 range. This is approximately 2 pH units below the pK a for the free product. The Raman spectrum of 4-dimethylaminobenzoyl-CoA undergoes major changes upon binding to dehalogenase. The bound form has two features near 1562 and 1529 cm -1 and therefore closely resembles the spectrum of product bound to wild-type enzyme, which underlines the quinonoid nature in these complexes. The use of a newly developed Raman system allowed us to obtain normal (nonresonance) Raman data for the dehalogenase complexes in the 100-300 µM range and heralds an important advance in the application of Raman spectroscopy to dilute solutions of macromolecules.

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“…As a consequence, max for the benzoyl chromophore shows only a modest red shift upon binding to the WT, from 260 to 304 nm (ε D 6.5 mM 1 cm 1 ). 12 Similar shifts are seen upon binding 4-MBA-CoA to W137F (ε D 6.5 mM 1 cm 1 ), W137A (ε D 14.1 mM 1 cm 1 ) and W137H (ε D 7.1 mM 1 cm 1 ). The Raman difference spectra for 4-MBA-CoA binding to WT and various Trp137 mutants are shown in Fig.…”

Section: Binding Of the Substrate Analog 4-methylbenzoylcoa To Dehalomentioning

confidence: 58%

“…The dissociation constants (K d ) of enzyme-4-MBA-CoA and enzyme-4-HBA-CoA complexes were measured by spectral titration as described previously. 4,12 …”

Section: Uv-visible Absorption Difference Spectral Analysis Of the Fomentioning

confidence: 99%

Raman evidence for product binding to the enzyme W137F 4‐chlorobenzoyl‐CoA dehalogenase in two conformational states

Dong¹,

Luo²,

Dunaway‐Mariano³

et al. 2005

J Raman Spectroscopy

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Part of the catalytic mechanism involves interaction between an active site side-chain, from Asp145, with the 4-position of the benzoyl moiety. For the complex consisting of product 4-HBA-CoA in the active site the interaction between Asp145 and the 4-OH group helps produce a major rearrangement in the benzoyl's p-electron system with the system becoming highly polarized. This leads to a large red shift in the benzoyl l max , from 260 to 370 nm, and radical changes in the Raman spectrum. In addition to the Asp145-4-OH interaction, the aspartate side-chain is held in place by a hydrogen bond to the indole side-chain of Trp137. We explored the consequences of removing this hydrogen bonding interaction by using protein engineering techniques to replace Trp137 by Phe, Ala or His. For 4-HBA-CoA binding to Trp137Phe dehalogenase there is spectroscopic evidence for two conformational populations for bound product. One species (highly polarized) has a l max near 375 nm and intense Raman bands at 1550, 1520 and 658 cm −1 , whereas the second species (moderately polarized) has a l max near 330 nm and an intense Raman mode at 1575 cm −1 . Raman data collected between 6 and 34 • C demonstrate population changes with the highly polarized species having the highest thermodynamic stability.

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Investigation of Substrate Activation by 4-Chlorobenzoyl-Coenzyme A Dehalogenase

Cited by 33 publications

References 29 publications

The active site dynamics of 4-chlorobenzoyl-CoA dehalogenase

The active site dynamics of 4-chlorobenzoyl-CoA dehalogenase

Modulating Electron Density in the Bound Product, 4-Hydroxybenzoyl-CoA, by Mutations in 4-Chlorobenzoyl-CoA Dehalogenase Near the 4-Hydroxy Group

Raman evidence for product binding to the enzyme W137F 4‐chlorobenzoyl‐CoA dehalogenase in two conformational states

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